www.nature.com/scientificreports
OPEN
miR-93 functions as an oncomiR for the downregulation of PDCD4 in gastric carcinoma
received: 21 July 2015
Hongwei Liang1,*, Feng Wang2,*, Danping Chu1,*, Weijie Zhang2,*, Zhicong Liao3,*, Zheng Fu1, Xin Yan4, Hao Zhu5, Wen Guo6, Yujing Zhang1, Wenxian Guan2 & Xi Chen1
accepted: 14 March 2016 Published: 29 March 2016
Programmed cell death 4 (PDCD4), as a tumor suppressor gene, is frequently reduced in a variety of tumors, including gastric cancer. Previous findings have indicated that PDCD4 participates in tumorigenesis through the regulation of apoptosis, but the molecular basis of this process has not been fully elucidated, and no studies have shown the upstream regulation of this gene in gastric cancer. In this study, we used bioinformatics analysis to search for miRNAs that could potentially target PDCD4 and identified miR-93 as a candidate. Moreover, we observed the inverse correlation between miR-93 and PDCD4 protein levels, but not mRNA levels, in human gastric cancer tissues. We further experimentally validated PDCD4 as the direct target of miR-93 by evaluating PDCD4 expression in gastric cancer cells after the overexpression or knockdown of miR-93. Additionally, the biological consequences of targeting PDCD4 through miR-93 were examined using cell apoptosis assays in vitro. We demonstrated that the repression of PDCD4 through miR-93 suppressed the apoptosis of gastric cancer cells. Finally, we revealed that miR-93 promoted the development of gastric tumor growth in xenograft mice by negatively regulating PDCD4. Taken together, the findings of the present study indicated the oncogenic role of miR-93 in gastric cancer tumorigenesis through targeting PDCD4, particularly in apoptosis. Gastric cancer is one of the most common human cancers. Although the incidence and mortality of this disease have decreased worldwide over the past 20 years, gastric cancer remains the fourth most common and the second most lethal cancer worldwide1. Knowledge of the molecular mechanisms underlying gastric cancer has major importance and might provide novel strategies to improve the survival and quality of life of gastric cancer patients. Therefore, it is important to understand the molecular basis of gastric cancer and explore new therapeutic agents. With substantial advances in our understanding of tumor biology, key oncogenes and tumor suppressor genes involved in mediating cancer growth and progression have become clear. These genes offer new targets for biological therapies. One such target is PDCD4 (programmed cell death 4). PDCD4 expression is commonly reduced in a variety of tumors2–5. As a tumor suppressor gene, PDCD4 is upregulated after the initiation of apoptosis6,7. In gastric cancer, PDCD4 regulates apoptosis through the downregulation of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis5. However, to the best of our knowledge, no studies have shown the upstream regulation of this gene in gastric cancer.
1
State Key Laboratory of Pharmaceutical Biotechnology, NJU Advanced Institute for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, School of Life Science, Nanjing University, Nanjing, Jiangsu 210093, China. 2Department of General Surgery, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 3Department of Cardio-Thoracic Surgery, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 4Department of Respiratory Medicine, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 5Department of Gastroenterology, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 6Department of Endocrinology, Nanjing Municipal Hospital for Governmental Organizations, Nanjing, Jiangsu 210018, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.Z. (email: yjzhang@nju. edu.cn) or W.G. (email: [email protected]) or X.C.(email: [email protected]) Scientific Reports | 6:23772 | DOI: 10.1038/srep23772
1
www.nature.com/scientificreports/ MicroRNAs (miRNAs) are small non-coding RNAs of 20 ~ 22 nucleotides. These molecules repress gene expression through interactions with the 3′ -untranslated regions (3′ -UTRs) of mRNAs. miRNAs target more than 50% of all human protein-coding genes, playing numerous regulatory roles in many physiological and developmental processes, including development, differentiation, apoptosis and proliferation8. miR-93 is one of the miRNAs often seen upregulated in cancers9–12. Several studies have reported the upregulation of miR-93 in gastric cancer13,14, showing that miR-93 functions as an oncomiR to enhance cancer cell apoptosis10,15–18. However, the molecular mechanisms underlying the contribution of miR-93 to the development and progression of gastric cancer remain elusive. In the present study, we identified PDCD4 as a direct target of miR-93. The potential role of miR-93 as an oncomiR of gastric cancer through PDCD4 targeting in apoptosis has been experimentally validated.
Materials and Methods
Cells and human tissues. The human gastric cancer cell lines AGS were purchased from the Shanghai
Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). AGS cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (GIBCO, CA, USA) and incubated in 5% CO2 at 37 °C in a water-saturated atmosphere. The gastric cancer and paired normal adjacent tissues were derived from patients undergoing a surgical procedure at the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China). All protocols concerning the use of patient samples in this study were approved by the Medical Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China). A signed consent form was obtained from each donor. The tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at − 80 °C. The clinical features of the patients are listed in Supplementary Table 1. Study protocol was approved by the Medical Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China) and all experiments were performed in accordance with approved guidelines of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China).
RNA isolation and quantitative RT-PCR. Total RNA was extracted from the cultured cells and human
tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Assays to quantify miRNAs were performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and a stem-loop RT primer (Applied Biosystems). The following reaction conditions were used: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. All reactions were run in triplicate. After the reaction, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT of the triplicate PCRs was determined. A comparative CT method was used to compare each condition with the controls. The relative levels of the miRNAs in cells and tissues were normalized to U6. The amount of miRNA relative to the internal control U6 was calculated using the 2−ΔΔCT equation, in which ΔΔCT = (CT miRNA − CT U6)target − (CT miRNA − CT U6)control. To quantify PDCD4 mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using oligo dT and AMV reverse transcriptase (TaKaRa), performed using the following conditions: 42 °C for 60 min and 70 °C for 10 min. Subsequently, real-time PCR was performed using the RT product, SYBRGreen Dye (Invitrogen), and specific primers for PDCD4 and GAPDH. The sequences of the primers were PDCD4 (sense): 5′- TATGATGTGGAGGAGGTGGATGTGA-3′; PDCD4 (antisense): 5′CCTTTCATCCAAAGGAAAAACTACAC-3′; GAPDH (sense): 5′-GATATTGTTGACATCAATGAC-3′; and GAPDH (antisense): 5′-TTGATTTTGGAGGGATCTCG-3′. The reactions were incubated at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec. After the reactions were complete, the CT values were determined using fixed threshold settings. The relative amount of PDCD4 mRNA was normalized to GAPDH.
Profiling of miRNA expression. The expression profiles of miRNAs in gastric cancer tissues and corresponding noncancerous tissues was determined using the miRCURY LNA Array system (version. 18.0, Exiqon Inc., Woburn, MA, USA) and conducted by KangChen Bio-tech, Inc. (Shanghai, China). RNA samples were labeled using the miRCURY Hy3/Hy5 Power labeling kit (Exiqon Inc., Woburn, MA, USA) and hybridized on the miRCURY LNA Array station. Scanning was performed with the Axon GenePix 4000B microarray scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). GenePix Pro version 6.0 was used to read the raw data of the images. The intensity of the signal was calculated after background subtraction, and replicated spots in the same image were averaged to obtain the median intensity. The median normalization method was used to obtain normalized data (foreground minus background divided by median). The significance of the results was determined using fold change and t-tests. The threshold value for significance used to define upregulation or downregulation of miRNAs was a fold change > 2 and p-value
OPEN
miR-93 functions as an oncomiR for the downregulation of PDCD4 in gastric carcinoma
received: 21 July 2015
Hongwei Liang1,*, Feng Wang2,*, Danping Chu1,*, Weijie Zhang2,*, Zhicong Liao3,*, Zheng Fu1, Xin Yan4, Hao Zhu5, Wen Guo6, Yujing Zhang1, Wenxian Guan2 & Xi Chen1
accepted: 14 March 2016 Published: 29 March 2016
Programmed cell death 4 (PDCD4), as a tumor suppressor gene, is frequently reduced in a variety of tumors, including gastric cancer. Previous findings have indicated that PDCD4 participates in tumorigenesis through the regulation of apoptosis, but the molecular basis of this process has not been fully elucidated, and no studies have shown the upstream regulation of this gene in gastric cancer. In this study, we used bioinformatics analysis to search for miRNAs that could potentially target PDCD4 and identified miR-93 as a candidate. Moreover, we observed the inverse correlation between miR-93 and PDCD4 protein levels, but not mRNA levels, in human gastric cancer tissues. We further experimentally validated PDCD4 as the direct target of miR-93 by evaluating PDCD4 expression in gastric cancer cells after the overexpression or knockdown of miR-93. Additionally, the biological consequences of targeting PDCD4 through miR-93 were examined using cell apoptosis assays in vitro. We demonstrated that the repression of PDCD4 through miR-93 suppressed the apoptosis of gastric cancer cells. Finally, we revealed that miR-93 promoted the development of gastric tumor growth in xenograft mice by negatively regulating PDCD4. Taken together, the findings of the present study indicated the oncogenic role of miR-93 in gastric cancer tumorigenesis through targeting PDCD4, particularly in apoptosis. Gastric cancer is one of the most common human cancers. Although the incidence and mortality of this disease have decreased worldwide over the past 20 years, gastric cancer remains the fourth most common and the second most lethal cancer worldwide1. Knowledge of the molecular mechanisms underlying gastric cancer has major importance and might provide novel strategies to improve the survival and quality of life of gastric cancer patients. Therefore, it is important to understand the molecular basis of gastric cancer and explore new therapeutic agents. With substantial advances in our understanding of tumor biology, key oncogenes and tumor suppressor genes involved in mediating cancer growth and progression have become clear. These genes offer new targets for biological therapies. One such target is PDCD4 (programmed cell death 4). PDCD4 expression is commonly reduced in a variety of tumors2–5. As a tumor suppressor gene, PDCD4 is upregulated after the initiation of apoptosis6,7. In gastric cancer, PDCD4 regulates apoptosis through the downregulation of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis5. However, to the best of our knowledge, no studies have shown the upstream regulation of this gene in gastric cancer.
1
State Key Laboratory of Pharmaceutical Biotechnology, NJU Advanced Institute for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, School of Life Science, Nanjing University, Nanjing, Jiangsu 210093, China. 2Department of General Surgery, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 3Department of Cardio-Thoracic Surgery, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 4Department of Respiratory Medicine, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 5Department of Gastroenterology, The Affiliated Drum Tower Hospital of Medical School of Nanjing University and Nanjing Multi-center Biobank, Nanjing, Jiangsu 210008, China. 6Department of Endocrinology, Nanjing Municipal Hospital for Governmental Organizations, Nanjing, Jiangsu 210018, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.Z. (email: yjzhang@nju. edu.cn) or W.G. (email: [email protected]) or X.C.(email: [email protected]) Scientific Reports | 6:23772 | DOI: 10.1038/srep23772
1
www.nature.com/scientificreports/ MicroRNAs (miRNAs) are small non-coding RNAs of 20 ~ 22 nucleotides. These molecules repress gene expression through interactions with the 3′ -untranslated regions (3′ -UTRs) of mRNAs. miRNAs target more than 50% of all human protein-coding genes, playing numerous regulatory roles in many physiological and developmental processes, including development, differentiation, apoptosis and proliferation8. miR-93 is one of the miRNAs often seen upregulated in cancers9–12. Several studies have reported the upregulation of miR-93 in gastric cancer13,14, showing that miR-93 functions as an oncomiR to enhance cancer cell apoptosis10,15–18. However, the molecular mechanisms underlying the contribution of miR-93 to the development and progression of gastric cancer remain elusive. In the present study, we identified PDCD4 as a direct target of miR-93. The potential role of miR-93 as an oncomiR of gastric cancer through PDCD4 targeting in apoptosis has been experimentally validated.
Materials and Methods
Cells and human tissues. The human gastric cancer cell lines AGS were purchased from the Shanghai
Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). AGS cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (GIBCO, CA, USA) and incubated in 5% CO2 at 37 °C in a water-saturated atmosphere. The gastric cancer and paired normal adjacent tissues were derived from patients undergoing a surgical procedure at the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China). All protocols concerning the use of patient samples in this study were approved by the Medical Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China). A signed consent form was obtained from each donor. The tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at − 80 °C. The clinical features of the patients are listed in Supplementary Table 1. Study protocol was approved by the Medical Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China) and all experiments were performed in accordance with approved guidelines of the Affiliated Drum Tower Hospital of Nanjing University (Nanjing, China).
RNA isolation and quantitative RT-PCR. Total RNA was extracted from the cultured cells and human
tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Assays to quantify miRNAs were performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and a stem-loop RT primer (Applied Biosystems). The following reaction conditions were used: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. All reactions were run in triplicate. After the reaction, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT of the triplicate PCRs was determined. A comparative CT method was used to compare each condition with the controls. The relative levels of the miRNAs in cells and tissues were normalized to U6. The amount of miRNA relative to the internal control U6 was calculated using the 2−ΔΔCT equation, in which ΔΔCT = (CT miRNA − CT U6)target − (CT miRNA − CT U6)control. To quantify PDCD4 mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using oligo dT and AMV reverse transcriptase (TaKaRa), performed using the following conditions: 42 °C for 60 min and 70 °C for 10 min. Subsequently, real-time PCR was performed using the RT product, SYBRGreen Dye (Invitrogen), and specific primers for PDCD4 and GAPDH. The sequences of the primers were PDCD4 (sense): 5′- TATGATGTGGAGGAGGTGGATGTGA-3′; PDCD4 (antisense): 5′CCTTTCATCCAAAGGAAAAACTACAC-3′; GAPDH (sense): 5′-GATATTGTTGACATCAATGAC-3′; and GAPDH (antisense): 5′-TTGATTTTGGAGGGATCTCG-3′. The reactions were incubated at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec. After the reactions were complete, the CT values were determined using fixed threshold settings. The relative amount of PDCD4 mRNA was normalized to GAPDH.
Profiling of miRNA expression. The expression profiles of miRNAs in gastric cancer tissues and corresponding noncancerous tissues was determined using the miRCURY LNA Array system (version. 18.0, Exiqon Inc., Woburn, MA, USA) and conducted by KangChen Bio-tech, Inc. (Shanghai, China). RNA samples were labeled using the miRCURY Hy3/Hy5 Power labeling kit (Exiqon Inc., Woburn, MA, USA) and hybridized on the miRCURY LNA Array station. Scanning was performed with the Axon GenePix 4000B microarray scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). GenePix Pro version 6.0 was used to read the raw data of the images. The intensity of the signal was calculated after background subtraction, and replicated spots in the same image were averaged to obtain the median intensity. The median normalization method was used to obtain normalized data (foreground minus background divided by median). The significance of the results was determined using fold change and t-tests. The threshold value for significance used to define upregulation or downregulation of miRNAs was a fold change > 2 and p-value
